This is an application to acquire a new patch-clamp electrophysiology setup. The complete setup consists of a fluorescent microscope, patch-clamp amplifier and a perfusion system. This equipment will primarily be used to study properties of the Mitochondrial Permeability Transition Pore (mPTP). mPTP is a large non-selective channel located in the mitochondrial inner membrane. It has been established that prolonged opening of mPTP during stress conditions leads to the increase in permeability of the mitochondrial membrane, disruption of energy generation in the form of ATP and eventually to cell death. mPTP opening is the central event leading to tissue damage during stroke. Thus, block of mPTP by pharmacological agents can be highly protective. However, current knowledge of the molecular composition of the channel (?pore?) part of mPTP remains incomplete. The central goal of the current proposal is to test the hypothesis that formation of the functional conducting channel of the mPTP requires the presence of non-proteinaceous polymers of inorganic polyphosphate and polyhydroxybutyrate in combination with the C-subunit and Ca2+. Experiments will involve use of several approaches. First, we will use electrophysiology to study the activity of the purified mPTP channel. Next, we will investigate the molecular composition and assembly of mPTP by using a number of analytical approaches including immunochemistry and mass spectroscopy. Finally, we will use wild-type and genetically modified cultured neurons and stable cell lines to investigate interactions between C-subunit, polyphosphate and polyhydroxybutyrate during mPTP activation in living cells. We expect that results of our study will lead to the detailed understanding of one of the most fundamental and critical molecular processes during cell death. In the future, this new knowledge will open opportunities for novel treatment strategies, which will specifically target these non-proteinaceous components of the pore, effectively prevent mPTP opening and protect against tissue damage.